A. Field of the Invention
The present invention relates to recombinant DNA technology. It is especially useful in allowing the introduction of foreign DNA into eukaryotic cells.
B. Description of the Art
There has been much interest in introducing foreign DNA into eukaryotic cells. One reaason for this interest is that some genetically caused diseases may be curable by introducing the foreign DNA into the cells, and allowing the foreign DNA to express a protein that the genetically defective cell cannot express. Another reason for this interest is that certain eukaryotic cells may prove to be the most suitable hosts for the production of certain eukaryotic proteins.
A very promising approach for achieving the introduction of foreign DNA into eukaryotic cells was disclosed in the article by K. Shimotohno and H. Temin, Formation Of Infectious Progeny Virus After Insertion Of Herpes Simplex Thymidine Kinase Gene Into DNA Of An Avian Retrovirus, 26 Cell 67-77 (1981). (The disclosure of this article and of all other articles cited in this application are incorporated by reference herein as if fully set forth).
This approach used retrovirus vectors for introducing foreign DNA into the vertebrate cell genome. Retroviruses are a family of RNA-containing viruses that replicate through a DNA intermediate. See generally H. Temin, Structure, Variation & Synthesis Of Retrovirus Long Terminal Repeat, 27 Cell 1-3 (1981)
In a normal life cycle, retroviruses integrate their DNA into the cell genome. It was discovered that it was possible to introduce the thymidine kinase (TK) gene of the herpes simplex virus type 1 into a retrovirus (spleen necrosis virus, SNV), propagate the recombinant virus to give an infectious virus, and then introduce the recombinant virus containing the foreign DNA into the cell genome. Through further research, it was discovered that other selected foreign genes (in addition to TK) could be inserted in a retrovirus vector. See K. Shimotohno and H. Temin, Loss Of Intervening Sequences In Genomic Mouse Alpha-Globin DNA Inserted In An Infectious Retrovirus Vector, 299 Nature 265-268 (1982).
However, in order to propagate a commercial quantity of recombinant retrovirus using this approach, one either had to construct the vector so as to make the virus replication competent, or one had to co-transfect the host cell where the virus was to be grown with a replication competent virus. Where the vector produces a replication competent virus, the resulting active virus will not be suitable for certain uses such as the introduction of the virus into a human body. If a replication competent virus is used for co-transfection, one must then be able to separate the two viruses after growth so as not to infect the human host with the second live virus after the stock has been produced. No satisfactory separation techniques are known to achieve this separation.
Thus, it can be seen that a need has existed for a relatively inexpensive way of producing a commercial size stock of replication incompetent retrovirus which contains a foreign gene and which is not contaminated by a replication competent second virus.